The ability of bacteria to survive antibiotic treatment remains a significant challenge for controlling bacterial infections worldwide. Unlike drug resistance where a mutation must evolve and be selected for, all microbial populations harbor persister cells which are a non-growing cell subpopulation highly tolerant to antibiotic action. These persister cells are a major cause of the multidrug tolerance of chronic infectious pathogens including tuberculosis (TB), the leading cause of bacterial mortality worldwide, as well as P. aeruginosa, and S. aureus. While much attention has been paid to antibiotic resistance, the mechanisms of antibiotic tolerance remain poorly understood despite their obvious importance for human health. We have identified ribosomal hibernation as a process that contributes to antibiotic tolerance in Listeria monocytogenes (Lm). During stress, Hibernation Promotion Factor (HPF), a conserved ribosome associated protein, is expressed and mediates dimerization of two 70S ribosomes into a translationally silent hibernating 100S ribosome. 100S ribosomes have been hypothesized to globally downregulate translation and promote stasis during stress, however, neither role has been experimentally demonstrated since their discovery nearly 30 years ago. Our work has demonstrated that 100S ribosomes are induced in response to treatment with aminoglycoside antibiotics and that deletion of HPF abolishes 100S ribosome formation and renders cells sensitive to these drugs. We hypothesize that ribosomal hibernation mediated by HPF alters translation in ways that contribute to antibiotic tolerance. The objective of this proposal is to characterize the role of ribosomal hibernation in antibiotic tolerance and to define the molecular mechanisms by which HPF-mediated 100S ribosomes facilitate persistence in Lm. In Aim1, we propose to use genome-wide analysis of in vivo translation to identify the genes and biological pathways regulated in response to ribosomal hibernation. In Aim 2, we will evaluate the mechanism(s) by which HPF-mediated hibernating ribosomes facilitate adaptive responses and promote antibiotic tolerance. These studies will be the first to describe a role for 100S ribosomes in antibiotic tolerance and lead to a better understanding of the mechanisms underpinning persistence. Since HPF is conserved in nearly every bacterial pathogen including those responsible for chronic infections like TB, understanding the role of HPF-mediated ribosomal hibernation in bacterial persistence has broad implications for human health.